Virulence and Immunomodulatory Roles of Bacterial Outer ... · Outer membrane (OM) vesicles are a naturally secreted product of Gram-negative bacteria. Vesicles form when a por-tion

MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, Mar. 2010, p. 81–94 Vol. 74, No. 11092-2172/10/$12.00 doi:10.1128/MMBR.00031-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Virulence and Immunomodulatory Roles ofBacterial Outer Membrane Vesicles

Terri N. Ellis and Meta J. Kuehn*Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

INTRODUCTION .........................................................................................................................................................81OUTER MEMBRANE VESICLE FORMATION BY PATHOGENS.....................................................................81

What Are OM Vesicles?...........................................................................................................................................81Natural OM Vesicles ................................................................................................................................................83Modulation of Vesiculation In Situ.........................................................................................................................83Vesiculation Observed during Infection ................................................................................................................84

ROLES OF OM VESICLES IN INTERBACTERIAL INTERACTIONS..............................................................85VESICLE-ASSOCIATED VIRULENCE FACTORS ................................................................................................85

Vesicle-Associated Toxins ........................................................................................................................................85Nontoxin Virulence Factors in OM Vesicles.........................................................................................................86Enrichment of Vesicle-Associated Virulence Factors...........................................................................................86

VESICLE INTERACTIONS WITH HOST CELLS .................................................................................................88Adherence of Vesicles to Host Cells.......................................................................................................................88Vesicle Entry into Cells............................................................................................................................................88Vesicle Fusion with Cell Membrane.......................................................................................................................89

IMMUNOMODULATORY ACTIVITIES..................................................................................................................89Vesicle-Mediated Activation of Inflammation and Innate Immunity ................................................................89LPS and Other PAMPs............................................................................................................................................90Vaccines, Antigenicity, and Adjuvanticity of OM Vesicles..................................................................................91

FUTURE DIRECTIONS ..............................................................................................................................................91Targeting Vesicles To Reduce Virulence................................................................................................................91Taking Advantage of OM Vesicles .........................................................................................................................91

ACKNOWLEDGMENTS .............................................................................................................................................91REFERENCES ..............................................................................................................................................................91

INTRODUCTION

Outer membrane (OM) vesicles are a naturally secretedproduct of Gram-negative bacteria. Vesicles form when a por-tion of the outer membrane with periplasmic content is selec-tively “blebbed” off to form round vesicles (11, 94, 95). Theproduction of OM vesicles has been observed for a wide varietyof gram-negative bacteria in all stages of growth as well as in avariety of growth environments, such as infected tissues. Theproduction of vesicles was further demonstrated to be linked tothe bacterial stress response (97). Vesiculation levels increaseduring periods of bacterial stress, such as what might be expe-rienced during the colonization of host tissues.

Analyses of OM vesicle components have demonstrated thatvesicles contain a wide variety of virulence factors (see refer-ences in Table 1). These virulence factors include protein ad-hesins, toxins, and enzymes as well as nonprotein antigens suchas lipopolysaccharide (LPS). Purified vesicles have displayedthe ability to act as a delivery system for virulence factors byinteracting with both prokaryotic and eukaryotic cells. Further-more, vesicles are laden with pathogen-associated molecularpatterns (PAMPs) and other OM components that can impact

the course of infection and host responses to infection. Thisreview will focus on examining the roles that OM vesicles andtheir specific components play as virulence factors and theirability to interact with and trigger responses from target cells.The diverse abilities of OM vesicles to modulate immune re-sponses, deliver toxins and other virulence factors to host cells,and aid in biofilm formation all attest to the importance thatthese secreted elements can have in bacterial pathogenesis.

OUTER MEMBRANE VESICLE FORMATIONBY PATHOGENS

An ever-growing number of pathogens have been docu-mented to produce and secrete natural OM vesicles (Table 1).Morphological and biochemical evidence for infected host tis-sues and fluids supports the idea that the production of vesiclesby pathogens occurs during infection and, in fact, may beinduced during infection.

What Are OM Vesicles?

OM vesicles are closed spheroid particles of a heteroge-neous size (�10 to 300 nm in diameter) released fromGram-negative bacteria during all phases of growth (11, 94,95). Electron microscopy (EM) studies reveal that OM ves-icles are formed from OM bulges and the subsequent fissionof vesicles containing electron-dense material (11, 18, 48,

* Corresponding author. Mailing address: Box 3711, BiochemistryDepartment, Duke University Medical Center, Durham, NC 27710.Phone: (919) 684-2545. Fax: (919) 684-8885. E-mail: [email protected].

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70, 81, 88, 94, 109). In general, OM vesicles reflect theOM composition, containing LPS, glycerophospholipids,and OM proteins as well as enclosed periplasmic compo-nents (43, 56, 61, 70, 95) (Fig. 1). Importantly, OM vesiclesare not a product of cell death since they contain newlysynthesized proteins and are produced without concomitantbacterial lysis (96, 99, 144). Several OM vesicle proteomeshave been evaluated recently, and all were determined to beenriched in envelope components, although some cytosolic and

inner membrane proteins were also present in these prepara-tions (10, 40, 84, 86, 87, 124, 141).

In addition to naturally shed vesicles, proteoliposomes canbe artificially generated by sonication or detergent treatmentof bacteria or the bacterial OM. These proteoliposomes areoften referred to as bacterial vesicles. In some cases, it wasclaimed that brief treatments of detergent or sonication simplyrelease preformed OM vesicles. However, in most cases, thesetreatments have been proven to differ in composition and ac-

TABLE 1. Virulence factors and activities associated with native OM vesiclesb

Species Vesicle-associated virulence factor(s) (reference�s�) Virulence-associated activity (reference�s�)a

Actinobacillus actinomycetemcomitans Leukotoxin, GroEL (46, 73) Bone-resorbing activity, chicken embryolethality, cytotoxic (22, 73, 105)

Actinobacillus pleuropneumoniae Apx toxin, proteases (102) Proteolytic (102)Bacteroides fragilis Hemagglutinin, alkaline phosphatase, esterase lipase,

acid phosphatase, phosphohydrolase, �- and �-galactosidases, �-glucosidase, glucosaminidase, �-glucuronidase (109)

Hemagglutinating and enzymaticactivities (109)

Bacteroides succinogenes Cellulase, xylanase (35) Aryl-�-glucosidase, aryl-�-xylosidase,endoglucanase, xylanase activities (35)

Bordetella pertussis AC-Hly, FHA, pertussis toxin (Ptx) (62) NDBorrelia burgdorferi OspA, OspB, OspD (26, 123) HUVEC adherence (123)Brucella melitensis Omp25, Omp31 (42) NDBurkholderia cepacia PLC-N, lipase, PSCP, 40-kDa protease (2) Enzyme activities (2)EHEC O111:H– ClyA (136) Pore forming (136)ETEC LT (61, 137, 138) Enterotoxic and vacuolating activities

(61, 78)ExPEC Alpha-hemolysin, CDT, iron and hemin binding OMPs

(6, 10)Hemolytic, causing detachment of cells

from monolayer (6)STEC O157:H7 Shiga toxin 2 (81, 143) Cytotoxic (143)UPEC CNF1 (83) Cytotoxic (83)Helicobacter pylori VacA, Lewis antigen LPS (34, 63, 75) Vacuolating activity (75), cytotoxic,

stimulating proliferation, IL-8secretion (66, 76)

Legionella pneumophila Mip (lpg0791), IcmK/IcmX, flagellin, phospholipase C,LaiE/LaiF, phospholipase, chitinase, acidphosphatases, Hsp60, proteases, diphosphohydrolase(40)

Inhibition of phagolysosome fusion,proteolytic and lipase activity (33, 40)

Moraxella catarrhalis UspA1/UspA2 (127) Binds C3 complement in serum (127)Neisseria meningitidis PorA, NlpB, NarE (putative) (93, 134) TNF-�, IL-6, activation of tissue factor

(procoaggulant), profibrinolytic andantifibrinolytic factors (12, 119)

Photorhabdus luminescens Toxin AB, GroEL (49) Insecticidal (79)Porphyromonas (Bacteroides)

gingivalisArg- and Lys-gingipain cysteine proteinases (27, 48, 72) Cleavage and loss of CD14 from

macrophage, cleavage of IgG, C3,IgM (47)

Pseudomonas aeruginosa Phospholipase C, hemolysin, alkaline phosphatase, Cif,PQS, quinolines, protease, �-lactamase (20, 21, 70,89, 91, 92, 119)

Decrease of apical CFTR expression, invitro enzyme activities, bactericidalquinolines, IL-8 stimulation (8, 20, 21,70, 91, 92)

Salmonella enterica serovarTyphimurium

Protective antigens (9) ND

Shigella dysenteriae serotype 1 Shiga toxin 1 (30) Toxicity (30)Shigella flexneri IpaB, IpaC, IpaD (68) Invasion (68)Treponema denticola Dentilysin, adhesins, proteases (19, 116) Chymotryptic activity, disruption of tight

junctions (19, 116)Vibrio anguillarum Metalloprotease, hemolysin, phospholipase (58) Protease, metalloprotease, hemolytic

activities (58)Vibrio cholerae RTX toxin (14) Cell rounding, depolymerizing actin (14)Xanthomonas campestris Cellulase, �-glucosidase, xylosidase, avirulence proteins,

type 3 secretion system proteins (124)ND

Xenorhabdus nematophilus Bacteriocin, fimbrial adhesin, pore-forming protein,chitinase (79)

Chitinase activity, insecticidal (79)

a In addition to proinflammatory properties of endotoxin and porins (130, 131).b Abbreviations not defined in the text: AC-Hly, adenylate cyclase-hemolysin; FHA, filamentous hemagglutinin; CDT cytolethal distending toxin; OMP, outer

membrane protein; PLC-N, phospholipase, nonhemolytic; UPEC, uropathogenic E. coli; CNF1, cytotoxic necrotizing factor 1; ND, not determined.

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tivity from naturally produced OM vesicles (12, 23). We havefocused this review of the literature on naturally produced OMvesicles.

Natural OM Vesicles

All Gram-negative bacteria investigated to date naturallyrelease OM vesicles. Calculations of native OM vesicle pro-duction show that the vesicles represent a significant fractionof cellular material. For instance, vesicles produced by typicallaboratory cultures of growing and dividing Pseudomonasaeruginosa and Escherichia coli cells account for �1% of theOM material in the culture (8, 43, 139). In contrast, Neisseriameningitidis produces abundant numbers of vesicles, constitut-ing 8 to 12% of radiolabeled protein and endotoxin in log-phase cultures (24). Not only are OM vesicles produced byfree-living cells, they are also abundant in naturally occurringbiofilms (120). In addition, intracellular pathogens such asLegionella pneumophila, Salmonella spp., and Francisella spp.produce OM vesicles in both intraphagosomal and extraphago-somal compartments (3, 33, 44, 45). In the case of Flavobac-terium, vesicles are made late in the growth phase (82). Themechanism of vesicle production is complex (95), and progressin this field will be updated in detail in a separate review (A.Kulp and M. J. Kuehn, unpublished data).

Modulation of Vesiculation In Situ

Rates of OM vesicle production are not uniform, even for aparticular strain, as production has long been seen to be influ-enced by environmental factors and by sources of cellularstress (74, 103, 129). In studies of both nonpathogenic andpathogenic species, vesiculation was found to be upregulatedby conditions that activate the �E envelope stress response(97). In fact, vesiculation appears to be critical to survivingstress. When vesiculation mutants of E. coli were challengedwith lethal envelope stressors, the vesicle-underproducing mu-tant succumbed, but the overproducing mutants survived bet-ter than the wild type (97). Considering the harsh antimicrobialenvironments encountered in a host during infection, the ca-pacity to modulate vesicle production is likely critical forpathogens.

EM evidence has shown that vesiculation can be induced byexposure to host components and tissue. In a model that sim-ulates meat spoilage, Pseudomonas fragi was inoculated intopig muscle (29). Unlike counterparts grown in a protein-freemedium, the surfaces of bacteria that contacted the meat werecovered with vesicles. These vesicles are thought to containproteolytic enzymes that disrupt myofibrils seen in the bacte-rial spoilage of meat. In addition, using a mouse model ofenterotoxigenic E. coli (ETEC) infection, vesicles were highly

FIG. 1. Model of OM vesicle production. Shown is the budding of the Gram-negative bacterial envelope. Released OM vesicles containperiplasmic material and OM proteins and lipids, including PAMPs and other virulence factors, as described in the text. Although details of themechanism remain unclear, budding is thought to occur in places where lipoprotein links between the OM and the peptidoglycan are broken ormissing.

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evident on ETEC cells recovered from the intestine 2 h afterintragastric inoculation (Fig. 2).

Antibiotic treatment has been demonstrated to influenceseveral aspects of vesiculation. The level of Shiga toxin-asso-ciated OM vesicle production by Shigella dysenteriae increasedwith mitomycin C treatment but not upon treatment with othertested antibiotics (30). Mitomycin C induces toxin production(135), and it is tempting to consider that one consequence ofincreased toxin production is hypervesiculation; however, thishas not been proven. Gentamicin treatment also increased thevesiculation of P. aeruginosa; however, when the compositionof these vesicles was inspected, they were not identical tonative OM vesicles, as they also contained inner membraneand cytoplasmic material (70). Nevertheless, it is important toconsider the tendency for particular antibacterials to elicit therelease of proinflammatory and toxic materials.

A careful biochemical analysis by Fernandez-Moreira et al.highlighted how naturally produced vesicles can mediate theOM surface remodeling that is critical for virulence (33). Theirdata demonstrated significant differences in the composition,LPS, and activity of OM vesicles shed by Legionella duringdevelopmentally distinct periods of growth. This work supportsa model by which transmissive Legionella pneumophila shedsOM vesicles into the phagosome not only to inhibit fusion withlysosomes but also to promote the remodeling of the bacterialsurface to the intracellular replicative form (33).

Vesiculation Observed during Infection

Examination of animal and human biopsy specimens, tis-sues, and host fluids provides evidence that bacterial vesiclesare formed during infection. EM has provided the most direct

observation of vesicle production by pathogens in host tissues.Some of the oldest evidence of bacterial OM vesicles comesfrom EM of gingival plaque, which demonstrates abundantmembrane vesicles interspersed between Gram-negative andGram-positive bacteria (50). This material, termed biofilms,has been the subject of much further study and is discussed inmore detail below.

Several biopsy studies have identified OM vesicles withinhuman tissues. EM examination of Helicobacter pylori-infectedhuman biopsy specimens revealed that bacterially derived ves-icles contact intestinal epithelial cells (34, 51, 63, 75). Hynes etal. further noted that these vesicles could adsorb antibodies insera from infected patients (63). EM examination revealed ahighly vesiculating Neisseria meningitidis serogroup B strainassociated with a fatal septic infection in a 20-year-old patient.The abundant “blebs” were thought to be the contributor tothe high endotoxin level of 1,700 endotoxin units/ml (equiva-lent to the activity of 170 ng/ml pure E. coli LPS) that led tothis fatal septicemia (101). Additionally, OM vesicles wereobserved in the cerebrospinal fluid of an infant infected with N.meningitidis and in the sera of three patients with lethal me-ningococcal endotoxemia (16, 125).

In several cases, microscopic examination of infected tissuesrevealed that OM vesicles are produced (or at least are moreapparent) near host cells. A nasal sample from a child withMoraxella catarrhalis sinusitis was examined by transmissionEM, and it was determined that, when in close association withleukocytes, M. catarrhalis secretes OM vesicles. Immunogoldlabeling of the C3 complement binding factor UspA1/UspA2showed that these proteins were located near or on OM vesi-cles in the biopsy specimen (127). In another study, a Salmo-nella strain isolated from a human food-poisoning infectionwas inoculated into the ileum of chickens. A subsequent EManalysis determined that a majority of the Salmonella cellsproximal to the ileal epithelial cells could be seen producingOM vesicles (142).

OM vesicles have also been observed at sites more dissem-inated from the direct site of bacterial colonization. OM ves-icle antigens were found in urine, blood, and several organs ofmice, dogs, and humans infected with Borrelia burgdorferi (26).Vesicles were observed on the surfaces of these spirochetesrecovered from infected ticks and from infected mouse urinarybladder, spleen, liver, heart, and brain tissues, indicating thatthese vesicles are formed by B. burgdorferi in vivo.

Even without direct observation by EM, OM vesicles havebeen implicated in the pathogenic process. In a series of stud-ies of E. coli infection, LPS and OM protein-containing bac-terial fragments were isolated from the serum of septic rats(52–54). E. coli cells grown in culture with serum were found toshed OM material with the same composition as the materialshed in vivo (55). Several factors indicate that these OM com-ponents are actually OM vesicles. They are composed of LPS,OM proteins, and lipoproteins; are larger than 0.1 �m in size;and are stable (55).

These studies leave no doubt that OM vesicles or blebs arefound surrounding and attached to the surface of pathogenicGram-negative organisms during infection. Subsequent studieshave been done to examine what roles they may play in thisniche and to address whether the vesicles are instruments ofvirulence and inflammation. It remains to be determined

FIG. 2. Enterotoxigenic E. coli recovered postinfection. Shown aredata from scanning electron microscopy of a representative ETECstrain recovered from mouse small intestines 2 h after intragastricinoculation. (Courtesy of Amanda McBroom.)



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whether OM vesicles contribute to the benefit of the bacterium(as a mediator of toxin transfer or inflammatory damage), tothe benefit of the host (as a mobile, potent indicator of infec-tion), or to both.

ROLES OF OM VESICLES IN INTERBACTERIALINTERACTIONS

The interaction of bacteria with cocolonizing and commen-sal bacteria can be hostile or neighborly. As discussed below,bacteria destroy coinfectors to prevent competition for limitednutrients or to provide nutrients for themselves. In other cases,vesicles facilitate bacterial communities where interspecies in-teractions are not only tolerated but are promoted in order tocombat otherwise lethal environmental conditions. In yet othercases, OM vesicles from one species have further been ob-served to aid in the survival of an entire mixed bacterial infec-tive population by actively destroying host defenses.

Vesiculating bacteria may have a survival advantage inmixed-population infections by their capacity to eliminatecompeting bacterial strains. As fuel for competition, OM ves-icles package periplasmic peptidoglycan hydrolases (70, 88).Their activity in OM vesicles has been attributed to the killingof cocultured Gram-negative as well as Gram-positive bacteria(89). Thus, OM vesicle production could be an advantage forgrowth in a mixed bacterial population where nutrition is lim-iting. Further, �-lactamase was found to be packaged into P.aeruginosa vesicles produced by strains that expressed �-lacta-mase (20). OM vesicles containing �-lactamase can protect thevesicle-producing strain from a cocolonizing, �-lactam-produc-ing species that could otherwise eliminate them. Additionally,some OM vesicles have been found to contain scavenging pro-teases (29, 129), xylanase, and cellulase (35), which can aid innutrient acquisition and thereby provide a survival advantage.

Vesicles can act as bridging factors in biofilms that producean environment that is resistant to antibiotics and antibacteri-als. Vesicles from oral bacteria, in particular, promote biofilmformation and colonization (48, 71, 72, 94). Vesicles are abun-dant in natural biofilms, and OM vesicle surface-bound DNAappears to be an electrostatic, bridging component that is cen-tral to membrane vesicle-bacterium-biofilm matrix interactions(120, 121). Porphyromonas gingivalis vesicles mediate the co-aggregation of the periodontopathogen Tannerella forsythia,and vesicles mediate the interaction/aggregation of staphylo-cocci and Prevotella intermedia (65, 71, 72). By participating inquorum sensing, OM vesicles can also aid survival by contrib-uting to communication within mixed populations of patho-gens. P. aeruginosa OM vesicles contain quorum-sensing mol-ecules, and vesicle production is also stimulated by theaddition of the Pseudomonas quinolone-signaling molecule,PQS (92).

Not only can OM vesicles provide interbacterial glue togenerate a nearly impervious multicellular structure, they canalso promote the growth of a cocolonizing pathogen. For in-stance, in a mixed population of bacteria, secreted vesicles thattitrate an antimicrobial agent or degrade �-lactams (describedabove) could significantly aid in the survival of any neighboringbacteria. Furthermore, OM vesicles released by one straincould cause increased inflammation, resulting in the exposureof host extracellular matrix proteins and the upregulation of

epithelial cell surface receptors that are beneficial to coloniza-tion by another strain. Moraxella catarrhalis is frequently foundin mixed infections with pathogens such as Haemophilus influ-enzae. As such, the ability of M. catarrhalis OM vesicles to bindcomplement and allow H. influenzae survival is a relationshipthat might also benefit M. catarrhalis (127). Thus, it was pro-posed that proinflammatory vesicle-mediated changes in hosttissue can pave the way for the adherence and survival ofcocolonizing pathogens.

Vesicles can also enable the exchange of bacterial products.Biochemical analyses revealed that DNase-resistant DNA anda periplasmic antibiotic could be transferred between bacteriaby OM vesicles, suggesting a fusion event (25, 67). In EMstudies where nonhydrolytic OM vesicles were used, P. aerugi-nosa and Shigella flexneri vesicles were found to confer stablyintegrated LPS onto the surfaces of other Gram-negative bac-teria (69). These results are particularly intriguing consideringthat they show a potential role for vesicles in natural transfor-mation and the acquisition of drug resistance in infections.

VESICLE-ASSOCIATED VIRULENCE FACTORS

Extracellular products of pathogens are often associatedwith acute infection and are essential for maximal virulence. Assecreted extracellular entities, OM vesicles can actively modifythe bacterial environment. OM vesicles have been found to becapable of delivering active toxins and other virulence-associ-ated bacterial proteins to host cells. In some cases, toxins areenriched in OM vesicles, suggesting that their entry into vesi-cles is somehow regulated.

Vesicle-Associated Toxins

Toxic materials are associated with native OM vesicles froma variety of Gram-negative pathogens. In many cases, vesicle-associated toxins have indeed been found to deliver activetoxins to host cells (Table 1). Enterotoxigenic E. coli (ETEC)produces heat-labile enterotoxin (LT) that associates with LPSin the particulate fraction of the cell culture supernatant (43,61, 137, 138). Purified ETEC vesicles specifically bind, enter,and deliver active toxin into epithelial and Y1 adrenal cells (61,78). OM vesicles from Actinobacillus actinomycetemcomitansexhibit cytotoxicity (22, 73, 105), and Xenorhabdus nematophi-lus and Photorhabdus luminescens OM vesicles were cytotoxicfor insect larvae as well as in tissue culture (79). Kolling andMatthews demonstrated, by immunoblotting, that Shiga toxinis present inside OM vesicles produced by O157:H7 cells (81),and this was corroborated for many strains of Shiga toxin-producing E. coli (STEC) under both aerobic and anaerobicconditions (143). NarE is a putatively vesicle-associated pro-tein of Neisseria meningitidis vesicles that exhibits ADP-ri-bosyltransferase and NAD-glycohydrolase activities typicallyassociated with toxicity (93). Toxic vesicles produced by ex-traintestinal E. coli (ExPEC) include a hemolysin; an RTXtoxin, which becomes surface bound; as well as cytolethal dis-tending toxin, which has a lipid binding domain to bind the OM(6, 10).

In some cases, vesicle-associated toxins have been observeddirectly in biopsy specimens of infected tissues. For instance,OM vesicles containing the vacuolating cytotoxin VacA were



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observed in H. pylori-colonized human gastric epithelium bi-opsy specimens and were similar in appearance and composi-tion to H. pylori vesicles made in vitro (34). The VacA-relatedtoxicity of H. pylori vesicles has been studied in some detail.OM vesicle-associated VacA causes vacuolization in HEP-2cells (75), although the relative contribution of vesicle-associ-ated VacA and free-soluble VacA is debated (114). In contrast,Galka et al. found that extracellular Mip was found exclusivelyin the OM vesicle fraction of Legionella pneumophila cultures(40).

H. pylori vesicles with either full-length VacA or a naturalcarboxyl-terminal truncate of VacA (from a VacA� phenotypestrain) induce cytochrome c-independent apoptosis in gastricepithelial cells (5). The truncated VacA contains the activedomain but not the type I secretion system signal; thus, theresults with the truncate were surprising because this was clas-sified as a “nonsecreted” version of VacA. It was proposed thatthe OM vesicles mediate the toxicity of the truncate by en-abling its presentation to host cells. This type of datum sug-gests that care should be taken when bacteria are assigned ashaving a toxin-deficient phenotype—they could be secretingtoxins via vesicles.

Investigations of several pathogens demonstrate directly orsupport that toxins are often localized to the surface of thevesicles. Extensive studies have detailed how LT is secreted,becomes associated with LPS, and eventually is shed via OMvesicles, which have LT tightly bound to LPS on their surfaceas well as soluble periplasmic LT in their lumen (59–61, 98)(Fig. 3). Although Kolling and Matthews demonstrated thatShiga toxin (Shiga toxin 1 and/or 2) is protease resistant in OMvesicles produced by O157:H7 (81), the ability of LPS to bindand neutralize Shiga toxin 2 (41) suggests that at least some ofthe Shiga toxin may be found on the OM vesicle surface in aprotease-resistant form. Immunogold EM was used to detect ahigh-molecular-mass protease on the surface of Actinobacilluspleuropneumoniae vesicles, which also package Apx toxin(102). The vacuolating toxin VacA was also seen to be closelyassociated with the membrane of H. pylori vesicles (75). VacAappears to be in a mature, monomeric form when associatedwith vesicles, as it is when it is associated with cells (64, 114).Studies of Campylobacter coli, C. fetus, and C. jejuni OM ves-icles showed that although present in both whole cells and OMvesicles, some proteins were not exposed to labeling in wholecells but were labeled in OM vesicles (90). This suggests thateither the topology changed (which is considered unlikely) orthe proteins internal in whole cells are externally associatedwith OM vesicles.

Toxins associated with the OM vesicle surface are typicallyvery protease resistant, although they are demonstrably on theexterior surface of vesicles. Leukotoxin detected on the surfaceof A. actinomycetemcomitans cells is found in a tight associa-tion with the membrane of its vesicles (73, 106). Similarly, thecytotoxins found to be associated with X. nematophilus OMvesicles were not strippable from OM vesicles except withsodium dodecyl sulfate, which destroyed its activity as well(79). LT and alpha-hemolysin (HlyA) were also tightly boundto the membrane of ETEC and ExPEC vesicles, respectively(6, 61). Whether the ability of toxins to tightly associate withthe vesicle surface is due to the high degree of curvature of the

OM vesicle or to their interaction with a specific component ofthe vesicles can be tested experimentally.

Nontoxin Virulence Factors in OM Vesicles

OM vesicles are a mechanism by which bacteria can secretemany periplasmic and membrane components associated withthe virulence of the bacterium. Often, OM components ofvesicles include adhesins that allow vesicles to interact withhost cells (Table 1) (discussed in more detail below). In addi-tion, OM vesicles can include proteases and signaling mole-cules. For example, Pseudomonas OM vesicles include viru-lence-associated enzymes, antimicrobial quinolines, andquorum-sensing molecules (70, 89, 91, 92, 113). Also, the P.aeruginosa CiF protein is preferentially secreted into vesicles,and CiF-containing vesicles decrease levels of apical lungepithelial expression of the cystic fibrosis transmembrane con-ductance regulator protein (CFTR), the central protein in thegenetic disease of cystic fibrosis (15, 91).

OM vesicle-associated proteins include active proteases thatcan degrade host cells. Vesicles from Treponema denticola, aperiodontopathogen, have active proteases that confer hostcell-damaging characteristics similar to those of the whole bac-terium (19, 116, 119). In addition, Neisseria meningitidis culturesupernatant endotoxic filtrates have procoagulant and fibrino-lytic factors for human monocytes (119).

OM vesicles can directly bind, titrate, and even destroy hostbactericidal factors. Indeed, Neisseria vesicles can bind andremove cell-targeted bactericidal factors in serum (110). H.pylori expresses Lewis antigens on its OM LPS, and this “mo-lecular mimicry” contributes to H. pylori virulence. Since OMvesicles produced by H. pylori contain those Lewis antigens ontheir LPS, it was proposed that these vesicles could contributeto the chronic stimulation of the host immune system (63).These vesicles can absorb anti-Lewis antigen autoantibodiesfrom the serum, and it was suggested that they may play a rolein the putative autoimmune aspects of H. pylori pathogenesis.M. catarrhalis OM vesicles carry UspA1 and UspA2, whichbind to C3 in the complement cascade (127). As a result, M.catarrhalis vesicles inhibit the bactericidal effects of comple-ment in normal human sera that are also directed againstnontypeable strains of H. influenzae. Thus, vesicles from onespecies can also contribute indirectly to the pathogenicity ofanother by binding and depleting complement in their imme-diate environment.

Additionally, OM vesicles can influence critical bacterialcell-host cell associations in a variety of tissues. P. gingivalisvesicles enhance the attachment and invasion of T. forsythia inperiodontal epithelial cells (65). Likewise, the addition of ad-herent-invasive E. coli (AIEC) vesicles to intestinal epithelialcells significantly increased the internalization of a vesicle-underproducing mutant of AIEC (115). The vesicle compo-nents that contribute to bacterial entry have yet to be deter-mined.

Enrichment of Vesicle-Associated Virulence Factors

Particular OM vesicle components may be enriched com-pared to their abundance in the originating bacterial cell.While vesicle OM protein profiles are typically very similar to



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FIG. 3. Models of binding, secretion, and vesicle-mediated LT transport into host cells. (A) LT consists of an LTA subunit in a complex with5 LTB subunits (LTAB5). LPS and GM1 both bind in interfaces between subunits of the LTAB5 complex, but the binding sites are distinct fromeach other. (B) LT is secreted through the inner membrane by the Sec machinery, folds into the periplasm, is secreted through the outer membranevia GspD of the type 2 secretory system, and binds to LPS on the cell surface via LTB5. Consequently, vesicles released from the cell have LT ontheir surface that can act as a tether between GM1 on the host cell and LPS on the vesicle. Vesicles are internalized by GM1- and cholesterol-richmicrodomains (lipid rafts). Vesicle-associated LT traffics through the Golgi apparatus and ER and is retrograde translocated into the cytosol,where the A1 fragment of LTA is catalytically active. The remaining vesicle material is maintained intracellularly in a nonacidified compartment.



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those of the OM, they are not identical. Enrichment and de-pletion of the OM and periplasmic cargo were found when thecontents of vesicles and bacterial OM fractions were compared(95). Most relevant to this discussion is the enrichment ofvirulence factors in OM vesicles. Proteins identified as beingenriched in vesicles include LT of ETEC and the P. aeruginosaaminopeptidase, both of which increase the association of ves-icles with cultured epithelial cells (7, 78). Leukotoxin and leu-kotoxic activities are 4- to 5-fold enriched in OM vesiclescompared with OM preparations for some strains of A. acti-nomycetemcomitans (73). Similarly, a subfraction of the vesi-cles secreted by ExPEC cells expressing HlyA is enriched inthat virulence factor (6). In contrast, CagA, an H. pylori viru-lence factor, is notable for its absence from VacA-containingOM vesicles from Cag pathogenicity island-positive strains(66, 75).

Quantitative proteomic techniques are useful tools to deter-mine exactly what is enriched in the OM vesicle population.When investigating the Xanthomonas campestris OM vesicleproteome, surprisingly, less than half of the most abundantOM proteins could also be identified in the vesicle fraction(124). Further analysis revealed that nearly half of the proteinsassociated with the OM vesicle fraction are involved or areputatively involved in virulence, and of the 21 proteins en-riched in the OM vesicle fraction, most of them are virulenceassociated. It was also evident that the composition of the OMvesicle proteins was partially determined by the growth me-dium. This indicates that in addition to their abundance, OMvesicle proteomes, including their associated virulence factorsand toxins, are highly mutable depending on the physiologicalsurroundings.

VESICLE INTERACTIONS WITH HOST CELLS

Protease- and toxin-containing OM vesicles interact withhost cells and thereby act as virulence factor delivery vehicles.As discussed below, their interaction can occur via a mem-brane fusion event or via adhesin-receptor-mediated attach-ment. In some cases, adherence is followed by vesicle uptake,even by nonphagocytic cell types. Data from diverse pathogenssupport the theory that vesicle binding contributes to infectionby enabling the delivery of toxic bacterial cargo to host cells,often by the internalization of the entire content of the vesicle.Thus, it is not surprising that, in addition to toxicity, the ram-ifications of such a delivery process include signaling and in-nate and adaptive immune responses in the host cells.

Adherence of Vesicles to Host Cells

Binding to cultured host cells has been observed for OMvesicles produced by a variety of pathogens. Protease- andtoxin-containing OM vesicles from E. coli, Shigella, Actinoba-cillus, and Borrelia strains interact with bacterial as well asmammalian cells (43, 68, 73, 117, 123). Vesicle-associated ami-nopeptidase increases the ability of P. aeruginosa vesicles toassociate with both primary and cultured human lung epi-thelial cells, although whether this is due to a receptorbinding or receptor-uncovering property of this enzyme isnot yet clear (7).

OM vesicles adhere to host cells not only in tissue culture

but also in the complex environment of an infected host tissue.For instance, H. pylori OM vesicles were bound to intestinalcells in biopsy specimens from infected patients (51, 75). Bac-teroides fragilis OM vesicles have hemagglutinating activity,which indicates that they are able to act as adhesive bridgingentities between mammalian cells (109).

Host cell adherence tropism depends on bacterial strain-specific factors. Not surprisingly, therefore, vesicles and bacte-ria can use identical host cell receptors. B. burgdorferi vesiclesadhere to human umbilical vein endothelial cells (HUVECs)in a manner that competes with whole Borrelia cells (140).Tropism in host cell binding was demonstrated for OM vesiclesfrom A. actinomycetemcomitans (22) and was specifically engi-neered for vesicles formed by E. coli that expressed the Yersiniaenterocolitica adhesin/invasin, Ail (77).

Vesicle Entry into Cells

In many cases, after OM vesicles adhere, they can be inter-nalized into host cells. Toxins can act as adhesins for the OMvesicles and allow vesicles to enter using the receptor-mediatedendocytic pathway also used by the soluble toxin. Topologi-cally, toxin-mediated vesicle adherence and uptake can occuronly if the toxin-vesicle interaction does not interfere with thetoxin-receptor interaction. This has been carefully mapped inthe case of LT, the toxin/adhesin associated with ETEC vesi-cles. Although the LT-B subunit forms a pentamer, and thusboth LPS and GM1 binding sites do not need to be simulta-neously occupied to confer vesicle binding, LPS (vesicle) andGM1 (host cell) binding sites on LT-B are entirely distinct (60,98) (Fig. 3A). As a result, ETEC vesicles bind to and enterepithelial cells via lipid raft-mediated endocytosis governed byGM1 (78) (Fig. 3B). Once inside the cell, the LT-A subunit istrafficked to the endoplasmic reticulum (ER) and the cytosol,where it catalyzes the ADP-ribosylation of Gs�, leading toenterotoxicity. The remaining vesicle components are stablymaintained in a nonacidified intracellular compartment.

Other examples of toxin-mediated vesicle adherence havebeen investigated. H. pylori VacA is taken up by gastric epi-thelial cells both as a free toxin and as a vesicle-associatedtoxin (34, 114). As yet, it is not known if VacA is the vesicleadhesin/entry ligand. Common components of vesicles such asOmpA could also contribute to the host cell entry of vesicles.OmpA mediates invasion for E. coli K1, causing neonatal men-ingitis, by interacting with a surface receptor, Ecgp, on brainmicrovascular endothelial cells (111, 112). Not all vesicle toxinsare required for effective vesicle adhesion. A. actinomycetem-comitans-derived OM vesicles, while highly enriched in leuko-toxin (73), do not require leukotoxin for rapid vesicle-cell in-teractions (22).

The destination of some vesicle-associated material is trans-epithelial. Dentilysin-carrying OM vesicles from Treponemadenticola can disrupt the epithelial cell monolayer in cultureand proceed to penetrate through the monolayer and emergeon the basolateral side, as measured by the appearance ofdentilysin activity in that compartment (19).

Host factors that contribute to vesicle uptake by specific hostcell types have been identified. For instance, bactericidal/per-meability-increasing protein (BPI), but not LPS binding pro-tein (LBP), enhanced the uptake of N. meningitidis vesicles



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into dendritic cells, and confocal imaging visualized BPI colo-calized with internalized OM vesicles (122). These interactionswere surprisingly selective, since the same did not occur formacrophages, and BPI did not promote similar interactions offree lipooligosaccharide aggregates with the dendritic cells.

Vesicle Fusion with Cell Membrane

Experimental evidence from several studies supports thehypothesis that certain OM vesicles can fuse with host cellplasma membranes. Salmonella cells growing within host cellvacuoles were found to release LPS that could be detectedwithin host epithelial cell vesicle membranes. This process wasdetermined to be dependent on the presence of the O antigenin the LPS structure, as detected by antibodies to the O antigenitself (44). According to another report, A. actinomycetem-comitans vesicles conferred a lipid-tracking dye to host cellplasma membranes within 2 min of coincubation (22). Like-wise, L. pneumophila OM vesicles tracked with a fluorescenttag were observed on the surface of alveolar epithelial cells,suggesting that they are lengthily adherent or have fused (40).

While these biochemical and visual data are compelling,abundant and definitive biophysical evidence of OM vesicle-membrane fusion is lacking. Meanwhile, it is intriguing toconsider how the phospholipid bilayer of mammalian cellscould possibly accommodate the foreign architecture of LPS,much less bacterial OM protein �-barrel structures (if theseare also transferred). The incorporation of such bacterial struc-tures into a eukaryotic membrane could also influence thesensing of the ligands by innate immune receptors and overallhost responses.

IMMUNOMODULATORY ACTIVITIES

The composition of OM vesicles makes them significantactivators of host innate and acquired immune response path-ways. In addition to the potent immunomodulatory moleculeLPS, vesicles contain OM porins and other important innateimmune-activating ligands. Together, vesicle components ap-pear to act synergistically to modulate the host response inways that can either stimulate the clearance of the pathogen,enhance the virulence of the infection, or both. In addition, theimmunogenic properties of OM vesicles lead to protectivemucosal and systemic bactericidal antibody responses thathave been exploited for vaccine purposes.

Vesicle-Mediated Activation of Inflammation andInnate Immunity

Vesicles are likely a key factor in effecting an inflammatoryresponse to pathogens. OM vesicles produced by colonizingbacteria encounter and may be taken up by epithelial cells andmacrophages to trigger an immediate innate host response.The response of epithelial cells and macrophages to secretedbacterial products is a well-established trigger of the inflam-matory cascade.

The ability of OM vesicles to trigger inflammatory responseswas thoroughly investigated by Alaniz et al. (1). Their analysisdemonstrated that OM vesicles from Salmonella enterica sero-var Typhimurium are potent stimulators of proinflammatory

cytokine secretion and immune cell activation. Salmonella OMvesicles activate macrophages and dendritic cells to increaselevels of surface major histocompatibility complex class II(MHC-II) expression as well as the production of the proin-flammatory mediators tumor necrosis factor alpha (TNF-�)and interleukin-12 (IL-12). OM vesicles also activated CD4 Tcells, indicating that protein antigen components of vesicleswere effectively processed and presented by the activated an-tigen-presenting cells.

A proinflammatory response to OM vesicles was also ob-served for several other pathogens. Studies of epithelial cellresponses found that H. pylori OM vesicles potently elicit anIL-8 response (66), as do P. aeruginosa vesicles (8). Detergent-generated vesicles from N. meningitidis, while not naturallyproduced, have well-characterized host responses due to theirdevelopment into a protective vaccine. These manufacturedvesicles have been shown to trigger the production of numer-ous proinflammatory cytokines from neutrophils, includingTNF-�, IL-1�, IL-8, macrophage inflammatory protein 1�(MIP-1�), and IP-10. (85) Naturally produced Neisseria OMvesicles were further found to stimulate IL-8, RANTES, andIP-10 while activating dendritic cell MHC-II expression (28).Likewise, OM vesicles purified from the fish pathogen Vibrioanguillarum stimulated the production of the proinflammatorycytokines TNF-�, IL-1�, and IL-6 when inoculated into floun-der (58).

It should be considered that stimulation by OM vesicles ismore likely to have a pathogen- and site-specific “signature”than is stimulation by individual purified components such asLPS or purified OM proteins. The protein composition ofvesicles can vary substantially with the bacterial strain of originand with the environment (87). Prominent OM proteins suchas porins are known to promote proinflammatory activities ina variety of Gram-negative pathogens, including H. pylori, Sal-monella, Fusobacterium, and Yersinia species (38, 39, 126, 130,131). Recently, the OM adhesin of N. meningitidis, NadA, wasshown to be required for the optimal activation of macrophageresponses. This adhesin fully activated macrophage cytokineproduction when presented in the conformation of the OMvesicle membrane rather than as a mixture of purified vesiclecomponents (128). Furthermore, the immunoglobulin re-sponses to the Neisseria antigen PorB delivered as a recombi-nant purified protein, as a virus-like particle, and in outermembrane vesicles were compared. Zhu et al. observed thatonly the OM vesicle delivery system generated a bactericidalserum response (145).

These data, as well as data from numerous vaccine studies(17, 104), demonstrate that the three-dimensional conforma-tion in which immune ligands are presented can greatly impactthe type and potency of the host response generated. Numer-ous studies have demonstrated that the composition of OMvesicles differs from that of the bacterial OM by the enrich-ment or exclusion of specific outer membrane proteins andLPS modifications (see above for a discussion of enrichment)(8, 70, 95). Given these differences in composition, it is hy-pothesized that the responses to these two bacterial mem-branes would be significantly different. Naturally producedOM vesicles and whole-cell OM preparations have not yetbeen compared in activation assays. Such experiments would



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help elucidate the specific roles that OM vesicles play in di-verting host responses to prevent bacterial clearance.

LPS and Other PAMPs

All experimental evidence to date indicates that the innateimmune response to OM vesicles results from the combinationof vesicle pathogen-associated molecular patterns (PAMPs)and LPS recognized in their natural context. Lipoproteins andOM proteins present in vesicles are biologically active mole-cules known to activate immune cells and induce leukocytemigration (140). The identity of PAMPs and other componentsof OM vesicles capable of modulating immune responses canperhaps best be investigated by studying the effects of OMvesicles from genetic mutants lacking specific envelope com-ponents.

The single most abundant, and typically considered mostpotent, immune-stimulating component of OM vesicles is LPS.LPS content can exceed the total protein content of vesicles byratios as high as 10:1 (our unpublished observations). Numer-ous studies have characterized how LPS is sensed by the Toll-like receptor 4 (TLR4) complex, triggering a proinflammatoryresponse common to the majority of Gram-negative bacterialinfections. High levels of LPS (endotoxin) and TLR4 activa-tion can lead to LPS toxicity and play a role in septic shock(107). Given the high LPS content, all investigations into im-mune responses to OM vesicles must define the contribution ofLPS to the host response. OM vesicles, as LPS delivery vehi-cles, have the capacity to either enhance bacterial clearance orcause host tissue damage by activating an inflammatory re-sponse.

Much of the research into the TLR4-mediated response toLPS has utilized pure, chemically extracted LPS. However, it ismore likely that LPS is naturally shed from bacteria in the formof OM vesicles. OM vesicles are heterogeneous proteolipo-somes that have a larger dimension than liposomes composedsolely of LPS (11), and these properties likely impact the typeand potency of the innate immune response. The predictedendotoxic capacity of vesicles has been established by numer-ous studies, but a more interesting question that has conse-quently arisen is whether vesicle-associated LPS is equally asendotoxic as cellular LPS.

To address this issue, Munford et al. compared the abilities ofphenol-extracted vesicle LPS and whole-cell LPS to bind to high-density lipoproteins (HDLs) (100). Those authors determinedthat purified and vesicle LPSs were similarly active in bindingHDL, while cell-associated LPS was less active. As a result, theyconcluded that vesicle-associated LPS is the native presentationof LPS that has the highest biological activity. In a similar com-parison, endotoxin secreted into the culture supernatant from N.meningitidis cells was found to more effectively stimulate mono-cyte expression of plasminogen activator and the procoagulantfactor TF than phenol-extracted, pure LPS (119).

Studies of LPS structure during the course of infection fur-ther demonstrated that LPS can be modified in response toenvironmental stimuli. LPS from P. aeruginosa has been welldocumented to exhibit an altered lipid A acylation patternwhen growing in the lungs of cystic fibrosis patients (32). Vari-ations of O-antigen composition have also been shown to varywith environmental conditions, as Sabra et al. were able to

induce P. aeruginosa to increase B-band LPS production underlow O2 conditions (116a). OM vesicles may also exhibit adistinctive population of LPS molecules, as an analysis of P.aeruginosa vesicles revealed vesicles enriched in B-band LPS(70). It is unclear if vesicles aid in the turnover of LPS mole-cules during colonization; however, the impact of these chem-ical alterations due to environmental conditions should befactored into the experimental design to determine host re-sponses to these LPS species.

The sensing of LPS by the TLR4 complex requires the ex-traction of LPS from the membrane by accessory proteins suchas CD14, LPS binding protein (LBP), or bactericidal/perme-ability-increasing protein (BPI). While the mechanism bywhich these factors remove LPS from the bacterial membranefor presentation to the TLR4 complex is not known, studieshave established OM vesicles both as key sources of LPS toactivate inflammation and as decoys, binding these solublefactors to impede inflammation.

Vesy et al. found that the LPS in Salmonella OM vesicleswas extracted by LBP but not by other factors such as CD14.The depletion of LBP from culture supernatants inhibited LPSbinding to cell surface TLR4-CD14 complexes (133). In a sim-ilar LPS binding study, Schultz et al. investigated the role ofBPI in binding Neisseria OM vesicle-derived LPS. Those au-thors observed that BPI binding of LPS was required for theOM vesicle interaction with dendritic cells, leading to CD14-mediated signaling and the expression of proinflammatory cy-tokines (122).

Vesicle-mediated components other than LPS have alsobeen shown to modulate the sensing of LPS by the host. Ves-icles from the dental pathogen P. gingivalis have been shown todecrease the level of membrane-bound expression of CD14 onmacrophage surfaces, leading to a decreased ability of themacrophages to trigger LPS-stimulated cytokine production. Aloss of CD14 is prevalent in cases of chronic periodontitis.Additionally, P. gingivalis OM vesicles were shown to bind andactively degrade soluble CD14 with vesicle-derived enzymes. P.gingivalis vesicles were also shown to degrade IgG, IgM, andcomplement factor C3 (47). These studies demonstrate thatLPS from OM vesicles both is directly sensed by the innateimmune system and directly interacts with host factors to mod-ulate that same response.

Purified LPS and vesicle-bound LPS not only differ in theirpotencies of stimulating an innate immune response but alsomay differ in their ability to distribute, and thus be cleared, inhost tissues. This is of particular importance for in vivo studiesthat instill pure LPS into animal models. The biophysical prop-erties of vesicles, as heterogeneous, proteinaceous, amphi-pathic structures, may allow greater movement through tissues.In contrast, pure LPS would likely exhibit more hydrophobicqualities, resulting in more rapid binding to available hostmembranes. As a result, vesicles could travel deeper into tis-sues where resident phagocytes are located. These same vesicleproperties may also result in vesicles being more readily rec-ognized and cleared by tissue phagocytes. Studies investigatingthe role of aggregate size in LPS binding by host cells supportthis hypothesis. Kitchens and Munford found that the size ofLPS complexes directly impacts the CD14-dependent internal-ization of LPS (80). Additionally, Elsbach noted that the in-flammatory responses to LPS are directly impacted by the



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presence of other membrane components as well as the mem-brane conformation (31).

OM vesicles have also been shown to contain other PAMPsin addition to LPS. P. aeruginosa vesicles were shown to con-tain both flagellin monomers and CpG DNA (8, 113). Studiesutilizing LPS-deficient OM vesicles from Neisseria demon-strated the ability of non-LPS PAMPs to enhance immuneresponses to vesicles (36, 108). However, to date, no studieshave demonstrated either flagellin or CpG DNA directly im-pacting the host responses to intact vesicles containing LPS.

Vaccines, Antigenicity, and Adjuvanticity of OM Vesicles

The capacity of OM vesicles to stimulate an adaptive mem-ory immune response has already been exploited in the devel-opment of effective OM vesicle-based acellular vaccines. Themost successful use of OM vesicles as a vaccine has been in thedevelopment of a vaccine against serogroup B N. meningitidis.The development and properties of these serogroup B vaccineshave recently been comprehensively reviewed by Holst andcolleagues (57). As detailed in that review, vesicle-based vac-cines have proven to be the only protective formulation againstserogroup B infections, with over 55 million doses adminis-tered to date. Several different formulations of the vaccineexist, targeted to strains and antigens specific to a given geo-graphic region. All preparations of Neisseria vesicle vaccinesstimulate protective mucosal and systemic bactericidal anti-body responses, with the antibody response being generatedpredominantly to the outer membrane porins PorA and PorB(37). Research is currently focused on engineering bacterialstrains to produce OM vesicles containing multiple PorA pro-teins derived from different strains in the hope of developing aglobal N. meningitidis serogroup B vaccine (132).

The concept of developing OM vesicle-based vaccines hasalso been investigated for cholera infections. Schild et al. usedthe neonatal mouse model of passive antibody transfer to dem-onstrate that OM vesicles from Vibrio cholerae generate aprotective antibody response (118). B-cell responses to OMvesicles from S. Typhimurium (1), B. burgdorferi (140), andFlavobacterium (4) have also been documented, indicating thatOM vesicles can easily be used as antigen delivery systems togenerate effective antibody responses.

The presence of LPS in OM vesicle-based vaccines has em-phasized the ability of LPS to act as a natural adjuvant to theimmune system. Neisseria is unusual in that it is a Gram-negative pathogen that can tolerate the deletion of the biosyn-thetic genes for LPS. However, immune responses to LPS-freeOM vesicle preparations are poor, and the addition of exoge-nous PAMP adjuvants was shown to be necessary to generatean optimal host response (28, 36, 108). The most commonlyused commercial OM vesicle vaccine against N. meningitidisutilizes manufactured vesicles from detergent-treated bacteria,which greatly reduces, but does not eliminate, the endotoxiccontent of vesicles to prevent toxicity. Data generated by uti-lizing LPS-deficient OM vesicles as well as the observation thatthe Neisseria adhesin NadA generates an effective immuneresponse only when presented with LPS indicate that the ad-juvant activity of LPS may be critical to the engineering of aneffective vaccine. Recent investigations into how TLR ligandsensing impacts other phagocyte functions such as phagosomal

maturation, the selection of antigens for MHC presentation,and dendritic cell maturation indicate that the immune systemmay be best adapted to sensing a “mixture” of LPS and otherOM bacterial components to generate an effective host re-sponse (13).

FUTURE DIRECTIONS

Since every pathogen expresses different virulence charac-teristics and utilizes these characteristics for distinct purposes,the quest to uncover the roles that OM vesicles play for everypathogen is daunting. Nevertheless, this substantial body ofresearch has generated a great deal of excitement, since it isrevealing both common and pathogen-specific roles that OMvesicles can play in bacterial virulence. Future work in this fieldis likely to take advantage of this knowledge in order to reducepathogenicity and to exploit the capacities of this complexnatural delivery mechanism.

Targeting Vesicles To Reduce Virulence

Disrupting the interactions between host cells and vesicle-associated virulence factors and between cocolonizing bacteriaand vesicles is likely to be a good strategy to reduce virulence.Preventing the generation of vesicles by pathogens and reduc-ing the proinflammatory effects of OM vesicles in host tissuesare also likely to reduce pathogenicity. As these processesbecome sufficiently understood, we can proceed to findingtherapeutics that can disarm vesicle effects and block vesicu-lation (if vesiculation is not an essential bacterial function) tomake the bacteria more susceptible to host defenses.

Taking Advantage of OM Vesicles

The utilization of OM vesicles as a complex of antigens in theirnative context with a natural adjuvant has already proven success-ful for human vaccines. Future efforts will likely result in OMvesicle vaccines engineered to reduce endotoxicity and to includemultispecies-specific antigens. Additionally, synthetic proteolipo-somes have been developed to deliver drugs to tumors and spe-cific tissues, although off-target effects are a continuing problem.Making use of adherence and entry mechanisms that target nativeOM vesicles to host cells could improve the specific targeting ofengineered therapeutic liposomes.

ACKNOWLEDGMENTS

We are grateful to Amanda McBroom for the postinfection image ofETEC obtained with the scanning electron microscope expertise ofLeslie Eibest in the Duke Biology Department.

This work was supported by a Burroughs Wellcome Fund Investi-gator in Pathogenesis of Infectious Disease award, NIH/NIAID grantsR01AI064464 and R01AI079068 (to M.J.K.), and a postdoctoral fel-lowship from The Hartwell Foundation (to T.N.E.).

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Virulence and Immunomodulatory Roles of Bacterial Outer ... · Outer membrane (OM) vesicles are a naturally secreted product of Gram-negative bacteria. Vesicles form when a por-tion